Heart failure results from impaired activation or deactivation of the heart at the level of the myofilament. Current dogma suggests that cardiac muscle contracts upon Ca2+ binding to cTnC, which regulates an on process in the thin filament (TF) leading to crossbridge (XB) attachment to generate force. Cardiac relaxation is regulated by a reverse off process in the TF triggered by rapid dissociation of Ca2+ from cTnC. It is thus believed that the kinetics of these structural changes modulate the kinetics of the XB cycle, such that pathology may arise from alterations in the relationship between the structural kinetics of the TF and XB cycling kinetics. However, previous ensemble studies failed to define the kinetic linkage between the TF processes and XB cycling. A main feature associated with TF regulation is Ca2+-induced dynamic interactions among the TF proteins, including multiple reversible structural changes at the TF protein interfaces. These forward and backward structural transitions represent the discreet signaling steps of the TF switching process that regulates XB cycling. Based on the findings from our recent in vitro dynamics study, we hypothesize that the microscopic kinetics of these forward and backward transitions in conformational state dictate equilibrium relationships between conformational populations and are tunable, and may thus provide the linkage between the rapid kinetics of Ca2+ exchange with cTnC and slow kinetics of XB cycling. However, the microscopic rate constants of individual steps cannot be easily determined by our current strategies that rely on ensemble-averaged measurements which obscure the spatial and temporal inhomogeneity of the protein dynamics present in the ensemble. Single-molecule spectroscopy has the unique advantage of unraveling this spatial and temporal heterogeneity inherent in ensemble samples. Accordingly, the overall objective of this project is to explore the use of single-molecule Forster Resonance Energy Transfer (smFRET) approaches to define the kinetic linkage between Ca2+-signaling and XB cycling by further characterizing the equilibrium relationships governing transitions between TF conformational populations. Importantly, microscopic forward or backward transition rate constants for each Ca2+-induced TF structural transition will be acquired. Two Specific Aims will be pursued using smFRET techniques to test our hypothesis: (1) examine the equilibrium relationships between conformational populations of cTnC at the level of single reconstituted regulatory units and (2) at the single-molecule level, determine microscopic rate constants associated with each Ca2+-induced reversible structural transitions of the C-domain of cTnI within single reconstituted regulatory units. Outcomes of this project will be of critical importane in addressing the current issue of the regulatory role of the TF in controlling XB cycling kinetics We expect that the information obtained from our proposed single-molecule studies will help to vertically advance the knowledge gained from our ensemble studies and muscle fiber research.